董春娟,徐勇勝
天津市兒童醫(yī)院呼吸科,天津 300074
血管新生是指在原有血管的基礎(chǔ)上新血管高度有序形成的過程。研究發(fā)現(xiàn)白血病患者骨髓微血管密度增加,說明血管新生也參與了白血病的發(fā)生。血管新生受多種細(xì)胞因子的調(diào)控,其中血管生成素(Angiopioetin, Ang)及其受體Tie2(tyrosine kinase receptor with immunoglobulin and epidermal growth factor homology domain-2)在腫瘤血管新生中具有調(diào)節(jié)血管穩(wěn)定、成熟和重建的作用。本文就近年Ang/Tie2在白血病中的研究進(jìn)展做一概述。
Ang及其受體Tie2是近年來發(fā)現(xiàn)的血管生成調(diào)節(jié)因子。Ang家族由血管生成素-1(Ang-1)、血管生成素-2(Ang-2)、血管生成素-3(Ang-3)、血管生成素-4(Ang-4)組成,與受體Tie2結(jié)合后參與血管新生。Ang-1主要由血管內(nèi)皮細(xì)胞(vascular endothelial cell,VEC)和管周細(xì)胞分泌,廣泛表達(dá)于全身各組織。Tie2為酪氨酸激酶受體,Ang-1與Tie2結(jié)合后引起酪氨酸激酶磷酸化,進(jìn)而發(fā)揮Ang-1維持新生血管穩(wěn)定,促進(jìn)新生血管成熟的作用[1]。Ang-2 亦由VEC和管周細(xì)胞分泌,在血管靜止區(qū)域幾乎不表達(dá),而在血管重建區(qū)(如胎兒肝臟、成人卵巢、子宮、腫瘤組織等)高表達(dá)。Ang-2是Ang-1的天然拮抗劑,能抑制Ang-1的功能,Ang-2 與Tie2結(jié)合后不能引起酪氨酸激酶磷酸化,從而導(dǎo)致血管退化[2]。另一方面,Ang-2能破壞VEC與細(xì)胞外基質(zhì)的相互作用,使不穩(wěn)定狀態(tài)的VEC對(duì)生長(zhǎng)因子的敏感性增加,從而加強(qiáng)了血管內(nèi)皮細(xì)胞生長(zhǎng)因子(vascular endothelial cell growth factor,VEGF)等細(xì)胞因子的促血管生成作用。因此,在僅有Ang-2存在時(shí),血管發(fā)生退化,血管數(shù)目減少,同時(shí)伴有VEC的凋亡和血管平滑肌細(xì)胞的斷裂;在Ang-2與VEGF同時(shí)存在時(shí),血管數(shù)目增加,VEC增生活躍,血管壁通透性增加[3]。然而在特定的組織微環(huán)境和實(shí)驗(yàn)條件下,Ang-2與Ang-1一樣起Tie2激動(dòng)劑的作用[4]。Ang-3為鼠源性血管生成素,Ang-4主要在肺內(nèi)表達(dá)[5]。酪氨酸激酶受體Tie2表達(dá)于內(nèi)皮細(xì)胞、部分造血干細(xì)胞、造血祖細(xì)胞及腫瘤細(xì)胞等多種細(xì)胞。Tie2在血管重建區(qū)和血管靜止區(qū)域均存在,說明其在血管新生和新血管穩(wěn)定中均發(fā)揮作用,且缺氧和炎癥性細(xì)胞因子都能誘導(dǎo)Tie2在VEC中表達(dá)[6]。研究發(fā)現(xiàn)Ang/Tie2在多種實(shí)體瘤中均表達(dá),并且與腫瘤的侵襲、預(yù)后等因素相關(guān)[3]。最近研究證實(shí),Ang/Tie2在白血病中亦表達(dá)并且與白血病的細(xì)胞形態(tài)、免疫分型、核型及預(yù)后等因素相關(guān)。
Ang及其受體在白血病中的表達(dá):Muller等[7]用半定量逆轉(zhuǎn)錄聚合酶鏈反應(yīng)(semiquantitative reverse transcription-polymerase chain reaction,RT-PCR)檢測(cè)了白血病細(xì)胞系和患者標(biāo)本中Ang-1、Tie2的mRNA水平,6種髓系白血病細(xì)胞系中4種高表達(dá)Ang-1;與正常對(duì)照相比,11例急性髓系白血病(acute myeloid leukemia,AMl)患者標(biāo)本中,9例高表達(dá)Ang-1,7例高表達(dá)Tie2;6例慢性粒細(xì)胞白血病(chronic myeloid leukemia,CML)患者標(biāo)本中,4例高表達(dá)Ang-1,6例高表達(dá)Tie2;所有外周血原始細(xì)胞大于20%的患者標(biāo)本中均能檢測(cè)到Ang-1的表達(dá),而5例完全緩解的髓系白血病,4例淋巴系白血病及正常對(duì)照均未檢測(cè)到Ang-1及其受體的表達(dá)。zhang等應(yīng)用RT-PCR方法檢測(cè)60例AMl患者外周血單個(gè)核細(xì)胞Ang-2表達(dá),發(fā)現(xiàn)Ang-2 mRNA表達(dá)水平明顯高于正常對(duì)照組[8]。
Ang及其受體與白血病細(xì)胞形態(tài)、核型及免疫分型的關(guān)系:Watarai等[9]用RT-PCR檢測(cè)了36例AMl患者Ang-1、Ang-2、Tie2的表達(dá),其中27例患者表達(dá)Ang-1、12例表達(dá)Ang-2、15例表達(dá)Tie2;Tie2在FAB分型的M2、M4、M5中高表達(dá),而在M1、M3中低表達(dá);Ang-2在CD7(+)的AMl患者中較CD7(-)的AMl患者表達(dá)高,而其他免疫表型如CD34、CD19、CD13、CD33均不影響Ang及其受體的表達(dá);Ang-2在伴t(8;21)的AMl患者中較不伴該染色體畸變的患者表達(dá)低。Riccioni等[10]檢測(cè)了111例AMl患者Tie2蛋白的表達(dá)情況,Tie2表達(dá)于59%的病例,其中22%的病例Tie2表達(dá)明顯增加;Tie2(+)的AMl患者大多表達(dá)單核細(xì)胞的膜標(biāo)記如CD14、CD11b、CD36,以及巨噬細(xì)胞集落刺激因子受體(macrophage colony-stimulating factor recepter,M-CSFR),F(xiàn)AB分型多為M4、M5a、M5b;Tie2(+)的AMl患者初診白細(xì)胞數(shù)較Tie2(-)的患者明顯升高,且fms樣酪氨酸激酶3(fms-like tyrosine kinase 3,F(xiàn)lt-3)表達(dá)高,突變率高;伴有inv(16)染色體異常的AMl患者大部分高表達(dá)Tie2。最近Hatfield等[11]培養(yǎng)91例AMl患者的原始細(xì)胞,其中62例患者細(xì)胞培養(yǎng)上清中檢測(cè)到Ang-1的表達(dá),23例患者檢測(cè)到Ang-2的表達(dá),進(jìn)一步研究發(fā)現(xiàn),伴有核磷蛋白(nucleophosmin1,NPM1)突變的細(xì)胞Ang-1表達(dá)明顯增加。綜上所述,Ang及其受體在M4、M5中表達(dá)高,并且與初診時(shí)高白細(xì)胞,t(8; 21)、inv(16)等核型異常,以及Flt-3、NPM1突變等相關(guān)。
Ang/Tie2在白血病中發(fā)揮作用的機(jī)制:Ang/Tie2在管新生中主要通過激活下游磷脂酰肌醇3激酶(phosphatidylinositol 3 kinase,PI3-K)途徑,活化絲氨酸-蘇氨酸激酶Akt,發(fā)揮抗凋亡作用, 促進(jìn)VEC生長(zhǎng)[12]。Ang/Tie2在白血病中是否也是通過PI3-K途徑發(fā)揮作用,不少學(xué)者對(duì)此進(jìn)行了研究。Watarai等[9]研究發(fā)現(xiàn)CD7(+)AMl細(xì)胞高表達(dá)Ang-2,且在人臍靜脈內(nèi)皮細(xì)胞(human umbilical vein endothelial cells)中存活時(shí)間長(zhǎng),因此推測(cè)Ang-2通過旁分泌作用于內(nèi)皮細(xì)胞上的受體,促進(jìn)血管新生來進(jìn)一步“飼養(yǎng)”白血病細(xì)胞。在白血病細(xì)胞培養(yǎng)液中,特別是Tie2(+)白血病細(xì)胞中能檢測(cè)到Ang-1的表達(dá),但白血病細(xì)胞與阻斷Ang-1/Tie2途徑的抑制劑共培養(yǎng)后,細(xì)胞凋亡增加,這表明Ang-1/Tie2能促進(jìn)白血病細(xì)胞的生長(zhǎng)[10]。Wakabayashi等[13]應(yīng)用Tie2-Fc段抑制Ang與Tie2受體結(jié)合,觀察Tie2-Fc對(duì)AMl細(xì)胞生長(zhǎng)的影響。19例患者標(biāo)本中,9例患者AMl細(xì)胞生長(zhǎng)未受Tie2-Fc的抑制,被歸為A組;10例患者AMl細(xì)胞生長(zhǎng)明顯受抑制,被歸為B組。同樣PI3-K抑制劑LY294002不能抑制A組AMl細(xì)胞生長(zhǎng),但能明顯抑制B組AMl細(xì)胞生長(zhǎng),而且LY294002和Tie2-Fc對(duì)AMl細(xì)胞生長(zhǎng)的抑制程度具有一致性。該結(jié)果也在細(xì)胞系中得到進(jìn)一步證實(shí),KG-1細(xì)胞的生長(zhǎng)能被Tie2-Fc和LY294002抑制,而HL-60細(xì)胞的生長(zhǎng)不受抑制。該實(shí)驗(yàn)說明,部分AMl細(xì)胞依賴Ang/Tie2生長(zhǎng),并且Ang/Tie2依賴下游的PI3-K途徑,促進(jìn)細(xì)胞生長(zhǎng),抑制凋亡。
Ang及其受體對(duì)白血病預(yù)后的意義:研究證實(shí),多種臨床或生物學(xué)因素如年齡、核型、Flt-3和NPM1等基因突變、對(duì)誘導(dǎo)治療的反應(yīng)等均影響AMl的預(yù)后[14]。近來Ang及其受體對(duì)白血病預(yù)后的影響引起了諸多研究者的興趣。Loges等[15]用實(shí)時(shí)定量聚合酶鏈反應(yīng)(real-time quantitative polymerase chain reaction,qPCR)檢測(cè)90例AMl患者外周血單個(gè)核細(xì)胞Ang-1、Ang-2、Tie2 和VEGF mRNA水平及其與預(yù)后的關(guān)系,多因素分析表明Ang-2及對(duì)誘導(dǎo)治療的反應(yīng)是總體生存率的獨(dú)立預(yù)后因素,高表達(dá)Ang-2者預(yù)后好,尤其是對(duì)VEGF低表達(dá)的患者。Schliemann等[16]用免疫組織化學(xué)法檢測(cè)AMl患者骨髓活檢標(biāo)本中Ang-1、Ang-2、Tie2蛋白的表達(dá),與正常對(duì)照相比,Ang-1表達(dá)無差別,Ang-2、Tie2表達(dá)增高;進(jìn)一步研究表明Ang-2高表達(dá)組總體生存率更高,特別是在Ang-1低表達(dá)組,Ang-2對(duì)預(yù)后的影響更明顯,而在Ang-1高表達(dá)組,Ang-2對(duì)預(yù)后的影響不明顯。然而,Hou等[17]用qPCR檢測(cè)126例AMl患者骨髓單個(gè)核細(xì)胞Ang-1、Ang-2、Tie2和VEGF的表達(dá),并分析各因素對(duì)預(yù)后的影響,多因素分析顯示Ang-2是總體生存率的獨(dú)立不良預(yù)后因素,特別是在中危核型組Ang-2對(duì)預(yù)后的影響顯著,而在低危和高危核型組則不明顯;且Ang-2對(duì)預(yù)后的影響在Ang-1或Tie2低表達(dá)、VEGF高表達(dá)患者中有意義。最近Kümpers等[18]分析了循環(huán)中Ang-2水平對(duì)高危AMl患者異基因造血干細(xì)胞移植后預(yù)后的影響,移植前循環(huán)中Ang-2高的白血病患者異基因造血干細(xì)胞移植后無病生存率低,且移植前Ang-2水平是異于其他已知因素(如骨髓浸潤(rùn)程度,移植前化療次數(shù))的獨(dú)立預(yù)后因素。Zhang等[8]應(yīng)用RT-PCR方法檢測(cè)AMl患者外周血單個(gè)核細(xì)胞Ang-1、Ang-2、Tie2和VEGF mRNA水平及其與預(yù)后的關(guān)系,發(fā)現(xiàn)Ang-2及是AMl患者總體生存率的獨(dú)立不良預(yù)后因素,而Ang-1和Tie2對(duì)總體生存率無影響。同時(shí)Koenecke等[19]發(fā)現(xiàn)移植前AMl患者血清中可溶性Tie2及Ang-2的水平較正常對(duì)照明顯增高,且可溶性Tie2為移植后復(fù)發(fā)的獨(dú)立危險(xiǎn)因素,特別是可溶性Tie2和Ang-2同時(shí)升高者總體生存率更低。Aref等[20]發(fā)現(xiàn)Ang-2高表達(dá)AMl患者總體生存率低,而Ang-2/Tie2比例是AMl患者獨(dú)立不良預(yù)后因素。
上述幾組實(shí)驗(yàn)結(jié)果截然相反,其原因一方面可能是患者樣本存在差異,另一方面可能由于所用實(shí)驗(yàn)技術(shù)不同。Loges等用qPCR檢測(cè)外周血原始細(xì)胞Ang/Tie2的表達(dá),Schliemann等用免疫組織化學(xué)法檢測(cè)骨髓活檢標(biāo)本,而Hou等用qPCR檢測(cè)骨髓單個(gè)核細(xì)胞,Kümpers等用酶聯(lián)免疫吸附反應(yīng)(enzyme linked immunosorbent assay,ELISA)檢測(cè)血清中的Ang-2。然而,從另一角度看相反的實(shí)驗(yàn)結(jié)果卻有共同之處,Loges等和Hou等的實(shí)驗(yàn)結(jié)果說明Ang和VEGF相互作用的重要性。如果將患者標(biāo)本分成VEGF高表達(dá)組和低表達(dá)組,Hou等的實(shí)驗(yàn)結(jié)果表明,在VEGF高表達(dá)組Ang-2是不良預(yù)后因素,而在VEGF低表達(dá)組Ang-2預(yù)后意義不顯著;Loges等的結(jié)果顯示,Ang-2和VEGF均高表達(dá)的患者比Ang-2高表達(dá)而VEGF低表達(dá)的患者預(yù)后差。由此可見,Ang-2和VEGF兩種細(xì)胞因子同時(shí)作用時(shí)患者預(yù)后較差[8,21]。
慢性淋巴細(xì)胞白血病(chronic lymphocytic leukemia,CLL)是一組有著不同病程的異質(zhì)性的疾病,多種預(yù)后因素決定CLL臨床病程和轉(zhuǎn)歸,免疫球蛋白重鏈可變區(qū)基因無突變(unmutated immunoglobulin heavy chain variable region,unmutated IgVH)、ZAP-70(+)(70-kDa zeta-associated protein)或CD38(+)均是獨(dú)立不良預(yù)后因素[22,23]。一些學(xué)者研究了Ang-2對(duì)CLL預(yù)后的意義,發(fā)現(xiàn)處于Binet分期B、C期的患者Ang-2表達(dá)高于A期患者,并且ZAP-70(+)CD38(+)的CLL患者比ZAP-70(-)CD38(-)的CLL患者高表達(dá)Ang-2[24]。另一些學(xué)者證實(shí),Ang-2高表達(dá)的CLL患者臨床分期較晚,CD38(+)和unmutated IgVH細(xì)胞百分比較高,不良預(yù)后核型較多見,無進(jìn)展生存期較短[25]。Binet A期的CLL患者中30%表達(dá)Ang-2,Ang-2的表達(dá)與骨髓微血管密度有顯著相關(guān)性;診斷時(shí)Ang-2高表達(dá)的患者疾病進(jìn)展快,需要早期治療,無進(jìn)展生存率低[26-28]。這些研究表明,Ang-2不僅反映了CLL腫瘤負(fù)荷,也反映了腫瘤的惡性程度,Ang-2高表達(dá)的患者惡性程度更高,進(jìn)展更快。
展望:Ang/Tie2在白血病中異常表達(dá),并且與白血病的細(xì)胞形態(tài)、免疫分型、核型及預(yù)后等因素相關(guān),是白血病治療的潛在靶點(diǎn)。臨床1、2期試驗(yàn)顯示,沙利度胺和兩種小分子酪氨酸激酶抑制劑(SU5416和SU11248)能明顯降低原發(fā)和復(fù)發(fā)難治白血病患者的骨髓微血管密度[29-31]。而Ang-2選擇性阻斷劑能顯著抑制鼠結(jié)腸癌標(biāo)本的血管新生[32]。最近,針對(duì)Ang的重組Fc片段在進(jìn)展期實(shí)體瘤中的臨床1期評(píng)價(jià)工作也已完成(www.clinicaltrials.gov/ct/show/NCT00102830)。綜上所述,研究Ang/tie2在白血病中的表達(dá)及其病理生理機(jī)制,將有利于白血病治療的進(jìn)展。
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